AmpliSens Legionella pneumophila-fep PCR kit

Transcription

1 For Professional Use Only AmpliSens Legionella pneumophila-fep PCR kit Instruction Manual AmpliSens Ecoli s.r.o., Studenohorska Bratislava 47 Slovak Republic Tel.: Fax: Federal Budget Institute of Science Central Research Institute for Epidemiology 3A Novogireevskaya Street Moscow Russia

2 TABLE OF CONTENTS 1. INTENDED USE PRINCIPLE OF PCR DETECTION CONTENT ADDITIONAL REQUIREMENTS GENERAL PRECAUTIONS SAMPLING AND HANDLING WORKING CONDITIONS PROTOCOL DATA ANALYSIS TROUBLESHOOTING TRANSPORTATION STABILITY AND STORAGE REFERENCES QUALITY CONTROL KEY TO SYMBOLS USED REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 2 of 16

3 1. INTENDED USE AmpliSens Legionella pneumophila-fep PCR kit is an in vitro nucleic acid amplification test for qualitative detection of Legionella pneumophila DNA in the clinical material (tracheal sputum or aspirate, nasopharyngeal and oropharyngeal swabs, bronchial washes or bronchoalveolar lavage, and autopsy material), microorganism cultures, environmental samples (water, washes from environmental objects, biofilms, and soil) using end-point hybridization-fluorescence detection of amplified products. The results of PCR analysis are taken into account in complex diagnostics of disease. 2. PRINCIPLE OF PCR DETECTION Legionella pneumophila detection by the polymerase chain reaction (PCR) is based on the amplification of pathogen genome specific region using specific Legionella pneumophila primers. In Fluorescent End-Point PCR, the amplified product is detected with the use of fluorescent dyes. These dyes are linked to oligonucleotide probes which bind specifically to the amplified product during thermocycling. A multi-channel rotor-type fluorometer is specially designed to detect fluorescent emission from the fluorophores in the reaction mixture after the PСR. It allows the detection of the accumulating product without reopening the reaction tubes after the PCR run. AmpliSens Legionella pneumophila-fep PCR kit uses hot-start, which greatly reduces the frequency of nonspecifically primed reactions. Hot-start is guaranteed by the separation of nucleotides and Taq-polymerase using a wax layer. Wax melts and reaction components mix only at 95 ºC. AmpliSens Legionella pneumophila-fep PCR kit can be used as: a qualitative test for Legionella pneumophila DNA detection in the clinical materials. During the test, multiplex end-point PCR of Legionella pneumophila mip-gene DNA and protrombin gene DNA is performed. Protrombin gene DNA is used as endogenous internal control. Legionella pneumophila mip-gene DNA amplification is detected in the channel for the JOE fluorophore, while the protrombin gene DNA amplification is detected in the channel for the FAM fluorophore. Protrombin gene DNA is a human genome DNA fragment; it should be present in an adequate amount in the DNA sample (no less than 10 3 genome equivalents). Both improper storage conditions and poor DNA extraction process can lead to DNA degradation and loss. So, the endogenous internal control allows not only to control analysis steps but also to estimate the adequacy of sampling and storage. REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 3 of 16

4 а qualitative test for Legionella pneumophila DNA detection in environmental samples. In this case the Internal Control STI-338 (IC) is used in the extraction procedure in order to control the extraction process of each individual sample and to identify possible reaction inhibition. Legionella pneumophila mip-gene DNA amplification is detected in the channel for the JOE fluorophore, while the Internal Control STI-338 (IC) DNA amplification is detected in the in the channel for the FAM fluorophore. 3. CONTENT AmpliSens Legionella pneumophila-fep PCR kit is produced in 2 forms: AmpliSens Legionella pneumophila-fep PCR kit variant FEP (0.5-ml tubes), REF B50-50-R0,5-FEP-CE. AmpliSens Legionella pneumophila-fep PCR kit variant FEP (0.2-ml tubes), REF B50-R0,2-FEP-CE. AmpliSens Legionella pneumophila-fep PCR kit includes: Reagent Description Volume, ml Quantity PCR-mix-1-FEP/FRT Legionella pneumophila ready-to-use singledose test tubes (under wax) colorless clear liquid tubes of 0.5 or 0.2 ml PCR-mix-2-FL colorless clear liquid tube PCR-mix-Background colorless clear liquid tube Mineral oil for PCR colorless viscous liquid dropper bottle DNA calibrator LS3 colorless clear liquid tubes Positive Control DNA Legionella pneumophila * colorless clear liquid tube DNA-buffer colorless clear liquid tube Negative Control (C-)** colorless clear liquid tubes Internal Control STI-338 (IC)*** colorless clear liquid tube * must be used in the extraction procedure as Positive Control of Extraction. ** must be used in the extraction procedure as Negative Control of Extraction. *** add 10 µl of Internal Control during the DNA extraction procedure directly to the sample/lysis mixture (see DNA-sorb-В, REF K CE protocol). AmpliSens Legionella pneumophila-fep PCR kit is intended for 55 reactions (including controls). 4. ADDITIONAL REQUIREMENTS Transport medium for storage and transportation of respiratory swabs. REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 4 of 16

5 0.9 % saline solution or 0.01 M potassium-phosphate buffer (ph 7.0). Reagent for pretreatment of viscous fluids (sputum, aspirates). DNA extraction kit. Disposable powder-free gloves. Pipettes (adjustable). Sterile pipette tips with aerosol filters (up to 200 µl). Tube racks. Vortex mixer. Desktop centrifuge with a rotor for 2-ml reaction tubes. PCR box. Personal thermocyclers (for example, Gradient Palm Cycler (Corbett Research, Australia). Fluorometer ALA-1/4 (Biosan, Latvia) or equivalent instrument. Refrigerator for 2 8 C. Deep-freezer at the temperature from minus 24 to minus 16 C. Reservoir for used tips. 5. GENERAL PRECAUTIONS The user should always pay attention to the following: Use sterile pipette tips with aerosol filters and use a new tip for every procedure. Store all extracted positive material (specimens, controls and amplicons) away from all other reagents and add it to the reaction mix in a distantly separated facility. Thaw all components thoroughly at room temperature before starting an assay. When thawed, mix the components and centrifuge briefly. Use disposable protective gloves and laboratory cloths, and protect eyes while samples and reagents handling. Thoroughly wash hands afterwards. Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in laboratory work areas. Do not use a kit after its expiration date. Dispose of all specimens and unused reagents in accordance with local regulations. Samples should be considered potentially infectious and handled in biological cabinet in compliance with appropriate biosafety practices. Clean and disinfect all samples or reagents spills using a disinfectant, such as 0.5 % sodium hypochlorite or another suitable disinfectant. REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 5 of 16

6 Avoid samples and reagents contact with the skin, eyes, and mucous membranes. If these solutions come into contact, rinse the injured area immediately with water and seek medical advice immediately. Safety Data Sheets (SDS) are available on request. Use of this product should be limited to personnel trained in DNA amplification techniques. Workflow in the laboratory must be one-directional, beginning in the Extraction Area and moving to the Amplification and Detection Area. Do not return samples, equipment and reagents in the area where the previous step was performed. Some components of this kit contain sodium azide as a preservative. Do not use metal tubing for reagent transfer. 6. SAMPLING AND HANDLING AmpliSens Legionella pneumophila-fep PCR kit is intended for analysis of the DNA extracted with DNA extraction kits from the clinical material (tracheal sputum or aspirate, nasopharyngeal and oropharyngeal swabs, bronchial washes or bronchoalveolar lavage, and autopsy material), microorganism cultures, and environmental samples (water, washes from environmental objects, biofilms, and soil). 6.1 Sampling Clinical material Tracheal sputum or aspirate should be taken to a disposable container. Nasopharyngeal swabs are obtained using sterile dry flocked swabs with plastic shafts for nasopharyngeal swabs. Gently insert the swab along the external nasal wall to a depth of 2 3 cm towards the inferior nasal concha. Then move the probe slightly lower, insert it in the inferior nasal meatus under the inferior nasal concha, rotate, and remove along the external nasal wall. When the material is obtained, insert the swab into a sterile disposable tube with 500 µl of Transport medium for storage and transportation of respiratory swabs ( REF 959-CE, REF 957-CE, REF 958-CE) or sterile saline or potassium-phosphate buffer solution. Break off the end of shaft or cut it off to allow tight closing of the tube cup. Close the tube with the solution and the swab. Oropharyngeal swabs are obtained using sterile dry rayon swabs with plastic shafts for oropharyngeal swabs. Rotate the swab over the surface of tonsils, palatine arches, and the posterior wall of the pharynx after gargling the oral cavity with water. When the material is obtained, insert the swab into a sterile disposable tube with 500 µl REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 6 of 16

7 of Transport medium for storage and transportation of respiratory swabs ( REF 959-CE, REF 957-CE, REF 958-CE) or sterile saline or potassium-phosphate buffer solution. Break off the end of shaft or cut it off to allow tight closing of the tube cup. Close the tube with the solution and the swab. It is recommended to combine nasopharyngeal and oropharyngeal swabs in a single tube. For this purpose, place the ends of both shafts into one tube containing 500 µl of Transport Medium for Storage and Transportation of Respiratory Swabs ( REF 959-CE, REF 957-CE, REF 958-CE) and analyze them as a single sample. Nasopharyngeal and oropharyngeal swabs are used for analysis in case of disease caused by L. pneumophila in form of acute respiratory disease (Pontiac fever). Bronchial washes (bronchoalveolar lavage) in disposable container. Autopsy material (fragments of affected parts of lungs). Microorganism cultures suspicious for Legionella spp. Resuspend cultures in 1 ml of saline or potassium-phosphate buffer, then centrifuge at 12,000 rpm for 15 min. The supernatant should be transferred into the disinfectant. Resuspend the sediment in 50 µl of saline solution. Use 50 µl of the suspension for DNA extraction. Store the above-mentioned material at 2 8 C for 1 day before the test, at the temperature below minus 16 С for 1 month and at the temperature below minus 68 C for 1 year. Only one freeze-thawing of the material is allowed. Environmental samples Water (wastewater, water from water bodies, and drinking water) (0.5 L) after pretreatment. Wipe samples from environmental objects are obtained using probe with a swab moistened in a sterile saline solution. The working part of the probe with the swab should be placed in a 1.5-ml tube with 0.5-ml of sterile saline solution. Break off the terminal part of the probe. 50 µl of the solution is used for DNA extraction. Biofilm scraped from internal surface of water-supply, industrial, and other types of equipment (for example, from trays in air conditioners). Samples of moist biofilms under water or at the water-air interface are obtained with a dry sterile probe (the working part of the probe with a swab is placed in a 1.5-ml tube with 0.5 ml of saline and the other part of probe is broken off and discarded). 50 µl of the sample is used for DNA extraction. Samples of dry biofilms are obtained using a swab saturated in sterile saline. The working part of probe with the swab is placed in a 1.5-ml tube with 0.5 ml of REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 7 of 16

8 sterile saline solution. Break off the terminal part of the probe. 50 µl of the sample is used for DNA extraction. Soil (100 g) is collected at sites of presumable bacterial contamination and used after pretreatment. Store the above-mentioned material at the temperature below 20 C for 1 week before the test, at the temperature below minus 16 С for 1 month and at the temperature below minus 68 C for 1 year. Only one freeze-thawing of the material is allowed. Temperature conditions for transportation are not limited. 6.2 Pretreatment The sample of bronchoalveolar lavage should be mixed by inverting in the initial vessel. Using a tip with aerosol filter, transfer 1.0 ml to a new tube and centrifuge it for 10 min at 10,000 rpm. Decant the supernatant leaving 100 µl of liquid above the pellet. Resuspend the pellet in 100 µl of supernatant and take 50 µl of the suspension for DNA extraction. The sputum should be treated with Mucolysin reagent REF 180-CE according to Mucolysin manual. Use 50 µl of the pretreated sputum for DNA extraction. If it is necessary to repeat the test, freeze the remaining sputum at the temperature below minus 16 С. Autopsy material is homogenized with a sterile porcelain mortar and pestle, with subsequent preparation of a 10% suspension in sterile saline or potassium-phosphate buffer. Transfer the suspension to a 1.5 ml tube and allow a precipitate to form for 1 3 min. 50 µl of the pretreated supernatant is used for DNA extraction. If it is necessary to repeat the test, store the remaining suspension frozen at the temperature below minus 16 С. Water samples. 0.5 L of water is preliminary filtered through a paper filter using a glass funnel. After preliminary filtration, water is filtered through a membrane filter with a pore diameter not more than 0.45 µm. After filtration, the membrane filter is cut with sterile scissors (to a disposable Petri dish) and placed with sterile pincers to 1.5-ml tubes with 1 ml of saline solution. The tube is incubated at room temperature for min under occasional mixing on vortex to ensure the transition of microflora to the solution. 50 µl of thus obtained solution is used for DNA extraction. The filtrate is to be stored at 2 8 C for 1 week. It can be frozen at the temperature below minus 16 С in case of longer storage. Soil. Transfer 0,4 1,0 g (~1.0 ml) of soil into the tubes with tightly close (screw) caps using individual spreader (or disposable paddle). Add 3 ml of saline solution to the each REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 8 of 16

9 tube, thoroughly mix them and decant for 5 min. The supernatant (50 µl) is to be used for subsequent study. 6.3 Disinfection: 1. Lysis Solution from DNA-sorb-В kit, REF K CE (if it has been stored at 2 8 С) should be heated at С until complete crystal dissolution. 2. Add 50 µl of Negative Control (C-) reagent to the pretreated samples (50 µl) and mix thoroughly. Then add 300 µl of Lysis Solution, heat at the temperature (65+1) С for 15 min. 7. WORKING CONDITIONS AmpliSens Legionella pneumophila-fep PCR kit should be used at C. 8. PROTOCOL 8.1. DNA extraction It is recommended to use the following nucleic acid extraction kit: DNA-sorb-В, REF K CE. The DNA extraction of each test sample (except for clinical material) is carried out in the presence of Internal Control STI-338 (IC). Addition of Internal Control STI-338 (IC) is not necessary for the samples of clinical material. Extract DNA according to the manufacturer s protocol taking into account next additions and improvements: Lysis Solution and Negative Control (C-) reagent have been already added to the tubes with test samples (see 6.3 Disinfection); Using tips with aerosol filter, add 10 μl of Internal Control STI-338 (IC) to the tubes with prepared environmental samples and microorganism cultures (see 6.3 Disinfection). Do not add Internal Control STI-338 (IC) to the tubes with clinical material (see 6.3 Disinfection). To prepare the Positive control of Extraction add 300 µl of Lysis Solution, 50 µl of Negative Control (C-) reagent, 10 µl of Internal Control STI-338 and 50 µl of Positive Control DNA Legionella pneumophila to the tube labeled PCE (Positive Control of Extraction); To prepare the Negative Control of Extraction, add 300 μl of Lysis Solution, 100 μl of Negative Control (C ) reagent and 10 µl of Internal Control STI-338 to the tube labeled C (Negative control of Extraction). After adding Universal Sorbent, Washing Solution 1, Washing Solution 2 and TE-buffer for DNA elution (after incubating at 65 C for 5 min), centrifuge samples at 8,000 10,000 rpm (10,000 13,000 rpm in case of using rotor with the 70 mm radius) each time. REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 9 of 16

10 8.2. Preparing PCR The total reaction volume is 25 µl, the volume of DNA sample is 10 µl. The type of tubes depends on the PCR instrument used for analysis Preparing tubes for PCR 1. Prepare the required number of the tubes with PCR-mix-1-FEP/FRT Legionella pneumophila and wax for the amplification of DNA from clinical and control samples. 2. Add 7 µl of PCR-mix-2-FL to the surface of the wax layer into each tube ensuring that it does not fall under the wax and mix with PCR-mix-1-FEP/FRT Legionella pneumophila. 3. Add above 1 drop of mineral oil for PCR (about 25 µl). 4. Prepare 2 tubes with PCR-mix-1-FEP/FRT Legionella pneumophila and mark them as Background. Add 17 µl of PCR-mix-Background to the surface of wax layer of each tube ensuring that it does not fall under the wax and mix with PCR-mix-1- FEP/FRT Legionella pneumophila. Add above 1 drop of mineral oil for PCR. 5. Using tips with aerosol filter, add 10 µl of DNA samples obtained at the DNA extraction stage. 6. Carry out the control amplification reactions: NCA Add 10 µl of DNA-buffer to the tube labeled NCA (Negative Control of Amplification). LS3 Add 10 µl of DNA calibrator LS3 to the tube labeled LS3 (Positive Control of Amplification). C Add 10 µl of the sample extracted from the Negative Control (C ) reagent to the tube labeled C (Negative control of Extraction). PCE Add 10 µl of the sample extracted from the Positive control DNA Legionella pneumophila reagent to the tube labeled PCE (Positive control of Extraction) Amplification 1. Run the following program on the thermocycler (see Table 1). 2. When the temperature reaches 95 C (pause mode), insert tubes into the wells of the thermocycler and press the button to continue. It is recommended to sediment drops from walls of tubes by short centrifugation (1 3 s) before placing them into the instrument. REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 10 of 16

11 Table 1 Amplification program Thermocyclers with active temperature adjustment: Thermocyclers with block temperature adjustment: MiniCycler, PTC-100 (MJ Research), Uno-2 (Biometra) GeneAmp PCR System 2400 (Applied Biosystems) GeneAmp PCR System 2700 (Applied Biosystems), MaxyGene (Axygen Scientific), Gradient Palm Cycler (Corbett research) Tempe- Tempe- Tempe- Step Time Cycles Time Cycles rapture, С rapture, С rapture, С Time Cycles 0 95 pause 95 pause 95 pause min min min s s s s s s s s s s s s s s s s s s min min min storage 10 storage 10 storage 3. Proceed to fluorescence detection after the amplification program is completed. 9. DATA ANALYSIS Please read ALA-1/4 Operating Manual before using this kit. The detection is performed by means of a fluorescence detector by measuring the fluorescence signal intensity in three channels: The channel for the FAM fluorophore (FAM channel or analogous, depending on the detector model) is intended for the detection of the signal of the IC DNA amplification product. The channel for the JOE fluorophore (HEX channel or analogous, depending on the detector model) is intended for the detection of the signal of the Legionella pneumophila DNA amplification product. Before the detection run, the required settings of the detector software should be adjusted according to the Guidelines [2]. The obtained results are interpreted on the basis of the level of fluorescence signal in the corresponding channels relatively to the background for the clinical and control samples. Interpretation is performed automatically by the software of the instrument used. The principle of interpretation is the following: Legionella pneumophila DNA is detected if the signal determined in the channel for the JOE fluorophore is greater than the specified threshold value of the positive result; Legionella pneumophila DNA is not detected if the signal determined in the channel REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 11 of 16

12 for the JOE fluorophore is less than the specified threshold value of the negative result, whereas the signal determined in the channel for the FAM fluorophore is greater than the specified threshold value. The result of the analysis is invalid if the signal determined in the channel for the JOE fluorophores is less than the specified threshold value of the negative result and signal determined in the channel for the FAM fluorophore is less than the specified threshold value. In such cases, the PCR analysis of this sample should be repeated starting from the DNA extraction stage. The result of analysis is equivocal, if the signal of a sample determined in the channel for the JOE fluorophore is greater than the specified threshold value of the negative result but less than the threshold of the positive result (the signal is between the threshold values). In such cases, the PCR of this sample should be repeated. If the negative result is obtained in the second run, the sample is considered equivocal and re-sampling is recommended. Threshold values are specified in the Guidelines [2] The result of the analysis is considered reliable only if the results obtained for the Positive and Negative Controls of amplification as well as for the Negative Control of extraction are correct (see Table 2) Results for controls Table 2 Control Stage for Fluorescent signal in the channel for the fluorophore control FAM JOE C DNA extraction > threshold value < threshold value of negative result PCE DNA extraction > threshold value > threshold value of positive result NCA PCR < threshold value < threshold value of negative result LS3 PCR > threshold value > threshold value of positive result 10. TROUBLESHOOTING The results of analysis are not taken into account in the following cases: 1. If the signal determined for the Positive Control of amplification (C+) in the FAM channel is less than the threshold value, this may indicate incorrect selection of amplification program and other mistakes of preparing PCR. Repeat the PCR once again. 2. If the signal determined for the Negative Control of extraction (C ) and/or Negative Control of amplification (NCA) in the FAM channel is greater than the threshold value, this indicates contamination of reagents or samples. In this case, the results of analysis REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 12 of 16

13 should be considered as invalid. The analysis must be repeated and measures for detecting of contamination source must be taken. If you have any further questions or if you encounter problems, please contact our Authorized representative in the European Community. 11. TRANSPORTATION AmpliSens Legionella pneumophila-fep PCR kit should be transported at 2 8 ºC for no longer than 5 days. 12. STABILITY AND STORAGE All components of the AmpliSens Legionella pneumophila-fep PCR kit are to be stored at 2 8 C when not in use. All components of the AmpliSens Legionella pneumophila-fep PCR kit are stable until the expiry date on the label. The shelf life of reagents before and after the first use is the same, unless otherwise stated. PCR-mix-1-FEP/FRT Legionella pneumophila is to be kept away from light. 13. SPECIFICATIONS Sensitivity Analytical sensitivity of AmpliSens Legionella pneumophila-fep PCR kit is not less than 1х10 3 copies per 1 ml of sample (copies/ml) Specificity The claimed analytical features of AmpliSens Legionella pneumophila-fep PCR kit are guaranteed only when additional reagent kit DNA-sorb-В (manufactured by Federal Budget Institute of Science Central Research Institute for Epidemiology ) is used. The analytical specificity of AmpliSens Legionella pneumophila-fep PCR kit is ensured by the selection of specific primers and probes as well as stringent reaction conditions. The primers and probes have been checked for possible homologies to all sequences published in gene banks by sequence comparison analysis. Specific activity of the kit was defined by investigation of the following strains from the American Type Culture Collection: Legionella pneumophila (serogroups 1-3: L.pneumophila Philadelphia 1 (ATCC 33152); L.pneumophila Togus 1 (ATCC 33154); L.pneumophila Bloomington (ATCC 33155)). Specificity was proved by the examination of the clinical material from true-negative (healthy) patients and the following cultures of microorganisms: 78 cultures of microorganisms from genus Bacillus, Citrobacter, Corynebacterium, Enterococcus, REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 13 of 16

14 Escherichia, Francisella, Klebsiella, Listeria, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus, Streptococcus, Yersinia and species of genus of Legionella (L.dumofii, L.longbeachae). In all cases the negative result has been obtained. The clinical specificity of AmpliSens Legionella pneumophila-fep PCR kit was confirmed in laboratory clinical trials. 14. REFERENCES 1. Handbook Sampling, Transportation, and Storage of Clinical Material for PCR Diagnostics developed by Federal Budget Institute of Science Central Research Institute for Epidemiology of Federal Service for Surveillance on Consumers Rights Protection and Human Well-Being, Moscow, Guidelines to the AmpliSens Legionella pneumophila-fep PCR kit for qualitative detection of Legionella pneumophila DNA in the clinical material, microorganism cultures, and environmental samples by polymerase chain reaction (PCR) with endpoint hybridization-fluorescence detection developed by Federal Budget Institute of Science Central Research Institute for Epidemiology. 15. QUALITY CONTROL In compliance with Federal Budget Institute of Science Central Research Institute for Epidemiology ISO Certified Quality Management System, each lot of AmpliSens Legionella pneumophila-fep PCR kit has been tested against predetermined specifications to ensure consistent product quality. REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 14 of 16

15 16. KEY TO SYMBOLS USED Catalogue number Caution Batch code Sufficient for In vitro diagnostic medical device Expiration Date Version Temperature limitation Manufacturer NCA Consult instructions for use Keep away from sunlight Negative control of amplification Date of manufacture C Negative control of extraction Authorised representative in the European Community LS3 Positive control of amplification PCE Positive control of extraction IC Internal control REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 15 of 16

16 VER RT ME РМ List of Changes Made in the Instruction Manual Location of Essence of changes changes Cover page The phrase For Professional Use Only was added The phrase The results of PCR analysis are taken into Intended use account in complex diagnostics of disease was added New sections Working Conditions and Transportation were added Content The Explanation of Symbols section was renamed to Key to Symbols Used The information about the shelf life of reagents before and after the first use was added Stability and Storage Information that PCR-mix-1-FEP/FRT Legionella pneumophila is kept away from light was added Key to Symbols Used The explanation of symbols was corrected Reference numbers were changed from B50-R0,5-FEP- Footer CE; B50-R0,2-FEP-CE to B50-50-R0,5-FEP-CE; B50-50-R0,2-FEP-CE The name of Institute was changed to Federal Budget Cover page, text Institute of Science Central Research Institute for Epidemiology Corrections according to the template and Russian Through the text Instruction manual 6.3 Disinfection The section was added 8.1 DNA extraction 9. Data analysis The sections were rewritten 10. Troubleshooting 13. Specifications 6. Sampling and handling Footer, 3.Content In the procedure of nasopharyngeal swabs sampling the probe with cotton swab was changed to flocked swabs with plastic shafts for nasopharyngeal swabs. In the procedure of oropharyngeal swabs sampling the probe with cotton swab was changed to rayon swabs with plastic shafts for oropharyngeal swabs REF B50-50-R0,2-FEP-CE was changed to REF B50-R0,2- FEP-CE REF B50-50-R0,5-FEP-CE; REF B50-R0,2-FEP-CE / VER / Page 16 of 16